Project Summary and Relevance Hearing loss is one of the most common forms of sensory deficits that affects hundreds of millions of people worldwide. Loss of the inner ear sensory cells, hair cells, is one of the major causes for deafness. Mammalian inner ear, unlike lower vertebrates, does not have the capacity to regenerate the lost hair cells or any inner ear cell types, which makes deafness a permanent deficit. With the exception of cochlear implantation, there is no medical treatment for deafness. Our immediate goal is to achieve hair cell regeneration in the mature mammalian inner ear and show such regeneration could lead to hearing recovery in animal models with hearing loss. Our long-term goal is to translate our work to human patients for restorative hearing therapies. One of the major challenges facing mammalian inner ear regeneration is the inability for the adult cochlear cells to be reprogrammed so they can proliferate and to respond to the signals and regenerate hair cells. We have made concerted efforts to conduct research targeting adult mammalian inner ear, by identifying the key pathways to reprogram and induce renewed proliferation and hair cell regeneration. By studying hair cell regeneration in the zebrafish model we identified c-Myc as a key gene in proliferation. By cutting-edge technologies, we uncovered the synergistic effect of co-activation of c-Myc and a potent inner ear progenitor gene Notch1, which reprograms adult and aged mouse cochlear cells for proliferation. Myc/Notch1 co-activation reprograms adult supporting cells to respond to the signals and transdifferentiate to hair cells in vivo. The proposal is a continued effort to expand on our original work utilizing Myc/Notch1 co-activation, with three new aims. First we will use a newly created transgenic mouse model to understand how to regenerate hair cells by Myc/Notch1 activation in the sensory epithelial region of adult cochlea efficiently in vivo. We will use Notch inhibition following Myc/Notch1 co-activation, for temporary Atoh1 induction in order to regenerate mature hair cells. We will test the application of direct delivery of MYC and NOTCH1 proteins to reprogram adult cochlea, which may lay a foundation for protein-based therapy in the future. Second, we will understand the pathways underlying Myc/Notch1 action by studying the involvement of mTOR pathway in reprogramming and using RNAseq to uncover the pathways Myc/Notch1 action, which should provide new routes for efficient hair cell regeneration. Third we will induce reprograming for proliferation and hair cell regeneration in mouse models with hearing loss induced by noise exposure, and to study the effect on hearing recovery. Considering the tremendous burden hearing loss brings to the individuals and society and the lack of progress in adult hair cell regeneration, our work of efficient hair cell regeneration in adult inner ear will have significant impact on the development of new treatment modalities aimed at eventual hearing restoration.